In the past we have used the proteasome inhibitor bortezomib to sensitize human tumor cell lines to apoptosis mediated by death ligands, in particular the protein TRAIL. Also we have performed studies with antibodies that act as agonists to the TRAIL death receptors. The agonist antibodies may offer specific advantages for cancer therapy since the specific agonist antibodies to the TRAIL death receptors may be more selective than the TRAIL protein itself that can bind to all of the receptors. More importantly, this combination of agents had an improved therapeutic effect over either agent alone in experimental lung metastases models of both the renal and breast carcinomas in vivo. In addition the therapeutic combination did not cause any obvious toxicity in the mice on examination of a large number of organs. This was the first demonstration, to our knowledge, that this combination could have therapeutic anticancer efficacy in vivo in the absence of significant toxicity. More recently, in collaboration with the Molecular Targeting Development Program (MTDP) of the NCI, we established a high-throughput screen to identify novel agents that can sensitize human cancer cells to TRAIL-mediated apoptosis. Over 50,000 pure synthetic compounds or natural products were screened and 18 compounds were identified for further study, 14 pure products and 4 synthetic compounds. Three of the compounds have been studied in more detail and none are proteasome inhibitors. We have identified two families of natural products, the cucurbitacins and the withanolides, where some family members sensitize tumor cells to TRAIL apoptosis whereas others do not. We recently published a manuscript on TRAIL sensitization by the cucurbitacins. Since the cucurbitacins are quite toxic, most of our current studies concern the withanolides. We have identified withanolide E as an extremely good sensitizer of various cancer cells to TRAIL-mediated apoptosis. It appears to sensitize the cancer cells by rapidly reducing the levels of the anti-apoptotic protein cFLIP. This is due to destabilization of the protein resulting in its degradation. However the exact molecular mechanism that results in increase cFLIP degradation is not clear an is currently under further investigation. Interestingly withanolide E can also sensitize cancer cells to the apoptotic effects of other TNF family death ligands. We established a collaboration with Professor Leslie Gunatilaka at the University of Arizona, a medicinal chemist and world expert on isolation, modification and biological properties of the withanolides. Testing of 80 analogues from Prof. Gunatilaka demonstrated that some withanolide analogues were 4-6 fold more potent than withanolide E for TRAIL sensitization. This analysis allowed for Structure Activity Relationship (SAR) studies that enabled identification of components of the withanolide E skeleton critical for this biological activity. Also we unexpectedly we observed that some of the active withanolides could also sensitize human melanoma cells to apoptosis in response to the viral mimetic and immune adjuvant poly (I:C). Additionally biotinylation of some active withanolides resulted in analogues with a very potent activity for the sensitization of melanoma cells to apoptosis. The molecular mechanism that underlies this activity is currently under investigation. Combinations of active withanolides plus poly (I:C) are currently been tested for therapeutic efficacy in a variety of mouse pre-clinical cancer models.